Implantable biological tissues can be formed of human tissues preserved by freezing (i.e., cryopreserving) the so called homograft tissues, or of animal tissues preserved by chemically fixing (i.e., tanning) the so called bioprosthesis (Carpentier, Biological Tissues in Heart Valve Replacement, Butterworth (1972), Ionescu, Ed.). The type of biological tissues used as bioprostheses include cardiac valves, blood vessels, skin, dura mater, pericardium, small intestinal submucosa (“SIS tissue”), ligaments and tendons. These biological tissues typically contain connective tissue proteins (i.e., collagen and elastin) that act as the supportive framework of the tissue. The pliability or rigidity of each biological tissue is largely determined by the relative amounts of collagen and elastin present within the tissue and/or by the physical structure and configuration of its connective tissue framework. Collagen is the most abundant connective tissue protein present in most tissues. Each collagen molecule is made up of three (3) polypeptide chains intertwined in a coiled helical configuration.
The techniques used for chemical fixation of biological tissues typically involve the exposure of the biological tissue to one or more chemical fixatives (i.e., tanning agents) that form cross-linkages between the polypeptide chains within a given collagen molecule (i.e., intramolecular crosslinkages), or between adjacent collagen molecules (i.e., intermolecular crosslinkages).
Examples of chemical fixative agents that have been utilized to cross-link collagenous biological tissues include: formaldehyde, glutaraldehyde, dialdehyde starch, hexamethylene diisocyanate and certain polyepoxy compounds. Of the various chemical fixatives available, glutaraldehyde has been the most widely used since the discovery of its antiimmunological and antidegenerative effects by Dr. Carpentier in 1968. See Carpentier, A., J. Thorac. Cardiovascular Surgery, 58: 467-69 (1969). In addition, glutaraldehyde is one of the most efficient sterilization agents. Glutaraldehyde is used as the fixative and the sterilant for many commercially available bioprosthetic products, such as porcine bioprosthetic heart valves (e.g., the Carpentier-Edwards™ stented porcine Bioprosthesis), bovine pericardial heart valves (e.g., Carpentier-Edwards™ Pericardial Bioprosthesis) and stentless porcine aortic valves (e.g., Edwards PRIMA Plus™ Stentless Aortic Bioprosthesis), all manufactured and sold by Edwards Lifesciences LLC, Irvine, Calif.
Fixation provides mechanical stabilization, for example, by preventing enzymatic degradation of the tissue. Glutaraldehyde has been extensively employed as a cross-linking agent to react with amino acid residues of collagen, such as the ε-amino groups of lysine and hydroxylysine or the carboxyl groups of aspartic acid and glutamic acid. The chemical nature of the glutaraldehyde-amine reaction is complex due to the reactivity of the glutaraldehyde molecule as well as the self-polymerization of dialdehydes. The most important component of the reaction products of an aldehyde and a primary amine involves the formation of a Schiff base wherein the nitrogen forms a double bond with the aldehyde carbon, replacing the double bond between the carbonyl carbon and the oxygen.
One problem associated with the implantation of many bioprosthetic materials is that the connective tissue proteins (i.e., collagen and elastin) within these materials can become calcified following implantation within the body. Such calcification can result in undesirable stiffening or degradation of the bioprosthesis. Two types of calcification—intrinsic and extrinsic—are known to occur in fixed collagenous bioprostheses. Intrinsic calcification follows the adsorption by the tissue of lipoproteins and calcium binding proteins. Extrinsic calcification follows the adhesion of cells (e.g., platelets) to the bioprosthesis and leads to the development of calcium phosphate-containing surface plaques on the bioprosthesis.
The factors that affect the rate at which fixed tissue bioprostheses undergo calcification have not been fully elucidated. However, factors thought to influence the rate of calcification include the patient's age, the existence of metabolic disorders (i.e., hypercalcemia, diabetes, etc.), dietary factors, the presence of infection, parenteral calcium administration, dehydration, in situ distortion of the bioprosthesis (e.g., mechanical stress), inadequate anticoagulation therapy during the initial period following surgical implantation and immunologic host-tissue responses.
In addition, glutaraldehyde fixation may have effect on tissue calcification. Further, in many cases, the fixed tissues are stored in media containing glutaraldehyde to maintain sterility. Unreacted glutaraldehyde or glutaraldehyde adsorbed during storage can leach out into the body post-implantation and cause side effects, as glutaraldehyde is suspected to be cytotoxic. In addition, unreacted aldehyde groups are typically present on the fixed tissue, which can become oxidized to carboxylic moieties. These moieties can attract calcium ions in vivo and contribute toward initiating calcification.
Efforts at retarding the calcification of bioprosthetic tissue have been numerous in recent years. The techniques resulting from these efforts may be broadly divided into two categories; those involving the pre- or post-treatment of glutaraldehyde-fixed tissue with one or more compounds that inhibit calcification (or modify the fixed tissue to be less prone to calcification) and those involving the fixation of the tissue with compounds other than glutaraldehyde, thereby reducing calcification.
The former category of techniques includes, but is not limited to, treatment with such compounds as: a) detergent or surfactant, after glutaraldehyde fixation; b) diphosphonates, covalently bound to the glutaraldehyde-fixed tissue or administered via injection to the recipient of the bioprosthesis or site-specifically delivered via an osmotic pump or controlled-release matrix; c) amino-substituted aliphatic functional acid, covalently bound after glutaraldehyde-fixation; d) sulfated polysaccharides, especially chondroitin sulfate, after glutaraldehyde fixation and preferably followed by treatment with other matrix-stabilizing materials; e) chitosan/heparin coupling after fixation; f) ferric or stannic salts, either before or after glutaraldehyde fixation; g) polymers, especially elastomeric polymers, incorporated into the glutaraldehyde-fixed tissue; or h) water-soluble solutions of a phosphate ester or a quaternary ammonium salt or a sulfated higher aliphatic alcohol, after glutaraldehyde-fixation.
The latter category of techniques for reducing the calcification of bioprosthetic tissue, i.e., techniques involving the fixation of the tissue with compounds other than glutaraldehyde, includes but is not limited to, the following: a) treatment by soaking the bioprosthetic tissue in an aqueous solution of high osmolality containing a photo-oxidative catalyst and then exposing said tissue to light thereby fixing the tissue via-photo-oxidization; and b) fixation via treatment with a polyepoxy compound, such as polyglycidyl ether (polyepoxy) compound.
Recently a new technique of calcium mitigation was described in U.S. Patent Publication No. 2003/0125813 A1, which is incorporated herein in its entirety. This method involves contacting fixed, unfixed or partially fixed tissue with a glutaraldehyde solution that has previously been heat-treated or pH adjusted prior to its contact with the tissue. Lee, et al. (J. Biomed. Mater. Res., 58(1);27-35 (2001)) have disclosed a method of mitigating unreacted glutaraldehyde residues by blocking with amino compounds, e.g., NH2—PEO—SO3 or heparin containing amino groups.
Although some of these techniques have proven to be efficient in reducing calcification, there remains a need in the art for further improvements of the existing techniques or for the development of new calcification-mitigating techniques to lessen the propensity for post-implantation calcification of fixed bioprosthetic tissues.